Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents.
Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents.
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DOI：
10.3389/fmicb.2017.01917
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发表时间：
2017
影响因子：
5.2
通讯作者：
Batoni G
中科院分区：
文献类型：
--
作者：
Grassi L;Di Luca M;Maisetta G;Rinaldi AC;Esin S;Trampuz A;Batoni G
持留细胞（PC）是常规细菌的休眠表型变体的子集，对抗生素具有高度耐受性。体内PC的产生可能解释了大多数慢性感染对抗菌治疗的抵抗力，以及鉴定能够靶向这些细胞的新抗菌剂的需求。本研究探讨了通过化学处理高效获得铜绿假单胞菌和金黄色葡萄球菌体外PC的可能性，并测试了它们对结构不同的抗菌肽（AMP）和两种临床使用的基于肽的抗生素，粘菌素和达托霉素的敏感性。这些分子的主要作用机制（即，膜扰动活性）使它们成为对抗休眠细胞的潜在候选物。暴露的固定相培养物的解偶联剂氰化物间氯苯腙（CCCP）的优化浓度能够产生高效率的PC表现出对不同类别的抗生素的耐药表型。CCCP处理的细菌的代谢谱进行了研究，通过监测细菌产热通过等温微量热法和通过评估氧化还原酶活性，流式细胞仪。CCCP预处理的细菌的两种细菌物种经历了显着减少产热和氧化还原酶活性，与未经处理的控制。去除CCCP后，诱导的持久性表现出延迟产热，与恢复正常生长前的滞后期相关。细菌的代谢再活化与其回复到对抗生素敏感的表型相一致。有趣的是，由CCCP处理产生的PC对三种不同的膜靶向AMP高度敏感，其水平与CCCP未处理的细菌相当。多粘菌素对铜绿假单胞菌的PC也有很高的活性，而达托霉素杀死了S。金黄色葡萄球菌的抗菌肽的浓度仅为所测试AMP的浓度的32至64倍。总之，CCCP处理被证明是以高效率产生医学上重要的细菌物种的体外PC的合适方法。重要的是，与传统抗生素不同，结构上不同的AMP能够根除PC，这表明此类分子可能代表开发针对持久性的新抗菌剂的有效模板。
Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.
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